Abstract

High glucose-induced oxidative stress is a major contributing mechanism to the development of diabetic cardiomyopathy. Nrf2 is an emerging critical regulator of cellular defense against oxidative damage. The role of Nrf2 in diabetic cardiomyopathy was investigated in vivo. Streptozotocin (STZ) induced diabetes in Nrf2 knockout (KO) mice that rapidly progressed to severe conditions with high mortality within two weeks of injection; whereas, in wild type (WT) mice, diabetes was less severe with no death. Severe myocardial lesions were observed in diabetic KO mice that had high, sublethal levels of blood glucose including: (a) irregular myocardial arrangements, myofibrillar discontinuation, and cell death; (b) reduced electron density, discontinuation of myocardial fibers, and mitochondrial damage; and (c) markedly reduced contractility of the cardiomyocytes to β-agonist stimulation. Parallel to severe cardiomyopathy, the diabetic KO hearts showed: (a) increased apoptosis as revealed by TUNEL and PARP1 cleavage assays; (b) infiltration of granulocytes and macrophages as well as fibrosis indicating robust inflammatory response; and (c) heightened oxidative stress as evidenced by increased levels of 8-hydroxydeoxyquanine, free malondialdehyde, and 3-nitrotyrosine. Increased oxidative stress in the KO hearts was attributed to decrease or loss of the basal and induced expression of Nrf2-dependent cytoprotective genes. Our findings demonstrate that loss of Nrf2 function synergizes with high glucose to cause heightened oxidative stress in the heart leading to severe diabetic cardiomyopathy.

Introduction

Diabetes mellitus is the world’s fastest growing disease with high
morbidity and mortality rates [1]. Epidemiological and experimental
studies have consistently reported diabetes as a strong risk factor for
the development of heart failure even after adjusting for concomitant
risks such as coronary artery disease and hypertension. This has
resulted in the recognition of a distinct disease process termed as
“diabetic cardiomyopathy” [2-4]. Factors influencing the pathogenesis
of diabetic cardiomyopathy remain unclear. It is generally believed that
high blood glucose promotes reactive oxygen species (ROS) production
resulting in oxidative stress in cells that in turn causes damage to
multiple tissues including the heart in diabetes [5-9]. On the other
hand, the repetitive contraction and high energy expenditure of the
heart expose it to excessive ROS production and make cardiomyocytes
vulnerable to oxidative damage [10]. Thus, a direct toxic effect of
hyperglycemia on myocardial tissues via ROS has been suggested as
a critical contributor to the development of diabetic cardiomyopathy.

The nuclear factor erythroid 2-related factor 2 (Nrf2) is a cap “n”
collar basic leucine zipper transcription factor that has recently emerged
as a critical regulator of the mammalian response to ROS, antioxidants,
and electrophilic signals [11-13]. Nrf2 controls the basal expression and
induction of a range of detoxification enzymes and antioxidant proteins
that coordinately metabolize toxic chemicals, scavenge ROS and repair
oxidative damage [11,14-17]. Genetic, animal, and epidemiological
studies have implicated Nrf2 in the development of a range of disease
and toxicity including cancer, chronic inflammation, and chronic
degenerative pathology. Nrf2-deficient mice have a tendency to develop
autoimmune lesions and a sponge-form leukoencephanopathy [18,19], as well as increased susceptibility to many toxicant-induced diseases,
such as benzo [α] pyrene-induced cancer [20], metal toxicity [21,22],
premature ovarian failure by 4-vinylcyclohexene diepoxide [23],
and bleomycin-induced airway inflammation and lung fibrosis [24].
Conversely, activation of Nrf2 by natural and synthetic antioxidants/
therapeutics has been shown effective for the prevention and treatment
of diseases and toxicities associated with oxidative stress [15].

Nrf2 is broadly expressed in animal tissues, but is rapidly degraded
through the ubiquitin-26S proteasome-mediated protein turnover.
Nrf2 and its binding protein Keap1 are redox sensors. Inducers, such
as phenolic antioxidants, Michael reaction acceptors, and transition
metals, modify critical cysteine residues in Keap1 and Nrf2 leading
to suppression of Nrf2 degradation and consequently, activation of
Nrf2 [25-28]. Activated Nrf2 translocates into the nucleus, dimerizes
with a small Maf protein, and binds to a common DNA sequence
called “antioxidant responsive element” (ARE) to up-regulate the
transcription of a battery of cytoprotective enzymes/proteins [17]. The
mechanism by which Nrf2 is activated endogenously under disease
conditions remains elusive. Presumably, endogenously produced
inducers, such as ROS and prostaglandins, mediate the activation of
Nrf2 [14].

We have previously shown that Nrf2 is critical in defense against
high glucose-induced oxidative damage in cultured cardiomyocytes [10]. In this study, we analyzed the role of Nrf2 in the development
of diabetic cardiomyopathy in vivo by using the Nrf2 knockout (KO)
mice and streptozotocin (STZ)-induced diabetic model. We found that
Nrf2 KO mice were highly sensitive to STZ-induced diabetic lesions
to develop severe cardiomyopathy as compared with wild type (WT)
control. Our findings revealed that loss of Nrf2 synergizes with high
glucose to induce a heightened oxidative condition in the heart of Nrf2
KO mice resulting in severe myocardial lesions.

Materials and Methods

Mice and treatments

Nrf2 knockout (KO) mice were derived by targeted gene
disruption as described elsewhere [29]. The mice were re-derived at
Jackson Laboratory to ascertain a specific pathogen-free status and
were backcrossed with C57BL/6J mice to obtain a C57BL/6 genetic
background (>97%) [30]. C57BL/6J mice from Jackson Laboratory
were used as controls. Mice were maintained at an environmentally
controlled National Institute for Occupational Safety and Health
animal facility accredited by the Association for Assessment and
Accreditation of Laboratory Animal Care International. The mice
were barrier maintained with a light/dark cycle of 12 hours at a
constant temperature (22°C) in HEPA-filtered, individually ventilated
microisolator cages (Thorn Caging Systems, Hazleton, PA) with
irradiated food (6% fat; 7913; Harlan-Teklad, Madison, WI). Water was
provided ad libitum. Sterile Beta Chips from Northeastern Products
Company (Warrensburg, NY) were used as bedding.

Eight-week-old male and female WT and Nrf2 KO mice on a
C57BL/6 background were given a single dose of streptozotocin
(STZ, 150 mg/kg body weight, i.p.; Sigma, St. Louise, MO) dissolved
in a sodium citrate buffer (0.1 M, pH 4.5); the control mice received
a single injection of the sodium citrate buffer only (8 mice/group/
gender). Whole blood glucose (WBG) from the mouse tail vain was
measured using the One Touch Ultra Blood Glucose Monitoring
System (Lifescan, Milpitas, CA) on the third day after STZ injection.
Mice with WBG levels of 250 mg/dL or higher were considered
diabetic. To exclude a potential direct effect of STZ on cardiotoxicity,
additional groups of WT and KO mice were included; the mice were
given STZ (4 mice/gender/genotype), and then insulin (Humulin U,
Eli Lilly, Indianapolis, IN) at a concentration of 10 units/mouse/day
immediately after hyperglycemia was diagnosed. Blood glucose levels
of the mice were monitored to maintain the levels between 100 and
200 mg/dL until sacrifice. Diabetic mice were observed for health daily.

Histopathology

Heart tissues were fixed and embedded in paraffin as described
previously [18]. Sections of 5 μm thickness were stained with
hematoxilin and eosin (H&E) and were examined under a light
microscope. Data were taken from representative cardiac sections from
three mice of each group (Control, STZ, and STZ+Insulin for each
gender and genotype); Magnification = 40 X.

Electron microscopy

Fresh heart samples were preserved in Karnovsky’s fixative, post
fixed in osmium tetroxide, mordanted in tannic acid, and stained en
bloc in uranyl acetate. The tissues were then dehydrated in alcohol and
embedded in Epon. Ultrathin sections were cut at 70 nm, collected
onto copper grids, and stained with uranyl acetate and lead citrate.
Ultrastructural sections were viewed and photographed using a JEOL JEM-1220 analytical transmission electron microscope (Tokyo, Japan).

TUNEL assay

Cardiac sections in paraffin were processed for TUNEL assay using
the DeadEnd Fluorometric TUNEL system (Promega, Madison, WI)
according to the manufacturer’s instructions. Briefly, the slides were deparaffinized,
re-hydrated, fixed with 4% formaldehyde, and permeated
with 20 μg/ml proteinase K and 0.2% Triton X-100 in PBS. The slides
were labeled with a TdT reaction mixture (Promega) for 90 min and
were mounted with a mounting solution containing 4’,6-diamidino-
2-phenylindole (DAPI) (Vectorshild, Vector Laboratories, Burigame,
CA). Fluorescence images of apoptotic cells (green) and cell nuclei
(blue) were obtained from a Zeiss LSM510 confocal miscroscope
with the fluorescein isothiocyanate (FITC)-DAPI setting. Fluorescent
pictures were taken with equal exposure times.

Detection of fibrosis

Cardiac sections of 5 μm thickness embedded in paraffin were deparaffinezed
and re-hydrated in distilled water. The slides were stained
with Picrosius Red (Direct Red 80, Fluka, Sigma, St Louise, MO) for
110 min, followed by dehydration as described elsewhere (www.cvm.
missouri.edu/vmdl/vmdl_his_SOP). Collagen fibers were stained red.
Images were taken using a light microscope with the SimplePCI 6
software (Compix Inc., Cranberry, PA).

Detection of granulocyte infiltration

The enzymatic activity of Naphthol AS-D chloroacetate esterase
was detected using the Naphthol AS-D chloroacetate esterase and
α-Naphthyl acetate esterase detection kit (Sigma) according to a
procedure from Sigma. Cardiac sections of 5 μm thickness in paraffin
were de-paraffinized and re-hydrated in distilled water. The slides were
stained with a freshly prepared Naphthol AS-D chloroacetate solution
at 37°C for 40 min. Esterase-positive granulation was shown on red and
brown under light microscope indicating infiltration of granulocytic
lineage cells in the tissue.

Immunoblotting

Hearts were taken from sacrificed mice, snap frozen in liquid
nitrogen, and stored at -80°C until use. Heart tissues were homogenated
with Ziroconia beads (BioSpec Products,Bartlesville, OK) and proteins
were extracted with the T-PER tissue protein extraction reagent
containing Halt protease inhibitors (Pierce, Rockford, IL). Extracts
of 60 μg protein each sample were separated on a 4-20% SDS-PAGE
gradient gel (Bio-Rad, Hercules, CA). Proteins separated were
transferred to PVDF membranes. The membranes were blocked with
5% non-fat milk for 1 h at a room temperature and blotted with specific
antibodies for overnight at 4oC with gentle shaking. After incubating
with appropriate horseradish peroxidase-conjugated secondary
antibodies, protein bands were visualized using ECL (Pierce). Anti-
PARP1 and anti-3-NT antibodies were obtained from Cell Signaling
(Beverly, MA).

Immunofluorescent staining

Cardiac sections in paraffin were deparaffinized, rehydrated, fixed
with 4% formaldehyde, and permeated with 20 μg/ml proteinase K
as described for TUNEL assay. The slides were then blocked with 5%
FBS in DMEM medium for 1 h and were incubated with anti-Mac-2
(macrophage antigen-2) (Cedarlane Laboratories Ltd., Burlington,
Ontariao, Canada) at a dilution of 1:500 or anti-8-OHdG(8-
hydroxydeoxyguanosine) (Japan Institute for the Control of Age, Fukuro, Japan) at a dilution of 1:100 in 2% FBS DMEM for 1 h at
room temperature. After washing with PBS for three times, the slides
were incubated with Alexa Fluor 488-conjugated secondary antibodies
(Invitrogen, Carlsbad, CA) at a dilution of 1:1000 in 2% FBS DMEM for
another hour. After washing for three times, the slides were mounted
with the DAPI mounting solution. Fluorescence images were obtained
under Zeiss LSM 510 confocal microscope with FITC-DAPI setting.

Lipid peroxidation

Free malondialdehyde (MDA) was measured using the Bioxytech
MDA-586 kit from OxisResearch (Potland, OR). Briefly, heart
tissues were homogenized in the presence of 5 mmol/L butylated
hydroxytoluene. Extracts were prepared by a centrifugation at 10,000 x
g for 10 min. MDA contents were determined spectrophotometrically
according to a procedure provided by the manufacturer.

Cardiomyocyte contractility

Adult mouse ventricular myocytes (AMVM) were isolated as
described below. Young adult mice (10 weeks old) were euthanized with
sodium pentobarbital and the heart was removed rapidly. The heart
was perfused with Krebs Hensleit Bicarbonate (KHB) at a constant rate
of 2 ml/min using a peristaltic pump. The heart was perfused with KHB
for 5 min, followed by changing to a low Ca++ KHB for an additional 10
min. The heart was then immersed in recirculating KHB with low Ca++ containing collagenase B for 30 min. The ventricle was minced and
placed into a 50-ml centrifuge tube, adjusted to 25 ml with low Ca++ KHB and centrifuged at 50 x g for 2 min, and the supernatant aspirated.
The concentration of Ca++ in KHB was increased in 3 increments (0.08,
0.6, 1.2 mM). Finally, the mixture was passed through a 225-μm nylon
mesh and centrifuged at 50 x g for 2 min. The centrifuge procedure
was repeated until the preparation was composed of 80% viable left
ventricular myocytes. Only those myocytes that were rod shaped, with
striations, no blebs and not spontaneously contracting were included
for analysis. Myocytes typically retained their baseline fractional
shortening for 4 h and were viable for biochemical and molecular
analyses for 24-48 h after harvest. Only freshly isolated cells were used
for physiologic experiments.

Measurements of the amplitude and velocity of unloaded single
AMVM shortening and relengthening were made on the stage of an
inverted phase-contrast microscope (Olympus, IX70- S1F2, Olympus
Optical Co., LTD., Japan) using the Myocyte Calcium Imaging/Cell
Length System in which the analog motion signal was digitized and
analyzed by the EDGACQ edge detection software (Ionoptix Cor.,
Milton, MA). Electrical field stimulation was applied at 1 Hz and about
20 volts to achieve threshold depolarization and experiments were
performed at 20% above threshold. Each cell serves as its own control
by continuous superfusion of buffers and drugs [31]. Data represent
the mean ± S.E.M. of 12-15 different determinations derived from 12-
15 different individual myocytes from seven to nine separate myocyte
preparations from seven to nine different mice.

Real-time PCR

Total RNA was reverse transcribed into single strand cDNAs, and
analyzed by real-time PCR using the SYBR GREEN PCR master mix
(Applied Biosystems, Foster City, CA) following standard procedures.
Briefly, single strand DNA template, forward and reverse primers (10
μM each), PCR master mix, and water were added to make a final volume
of 50 μl. Thermal cycling was carried out as follows: 95°C for 3
min as initial denaturing, followed by 45 cycles of 94°C for 30 sec, 60°C
for 30 sec, and 72°C for 60 sec, and a final extension at 72°C for 2 min. Threshold cycles (CT values) were determined. Real-time PCR results
were normalized using 1% of input as an internal control. Relative DNA
amounts were calculated from CT values for each sample by interpolating
into the standard curve obtained using a series of dilutions of standard
DNA samples that were run under the same conditions. The sequences
of the primer sets used for real-time PCR are as follows: Nrf2, forward:
5’-AGGAAGCTGGAGAACATT-3’; reverse: 5’-GTTTTTCTTTGTATCTGG-
3’. Mouse NQO1: forward, 5’-CCCCACTCTATTTTGCTCCA-
3’; reverse, 5’-ACTCCTTTTCCCATCCTCGT-3’. Actin:
forward, 5’-GACCTCTATGCCAACACAGT-3’; reverse, 5’-ACTCATCGTACTCCTGCTTG-
3’. Representative data from three separate
experiments were presented.

Statistical analysis

Data were collected from repeated experiments and were
represented as means ± standard deviations. One-way ANOVA and
Student’s t test were used for statistical analysis. A p value of <0.05 was
considered statistically significant.

Results

Severe cardiomyopathy in diabetic Nrf2 KO mice

We have previously reported that Nrf2 is critical in defense against
high glucose-induced oxidative damage in cultured cardiomyocytes,
supporting the notions that diabetic cardiomyopathy results from
the exposure of cardiomyocytes to high levels of blood glucose and
Nrf2 has a protective role against diabetic myocardial lesions by
suppressing oxidative damage [10]. In the present study, we examined
if Nrf2 protects against diabetic cardiomyopathy in intact animals by
comparing diabetic lesions between Nrf2 WT and KO mice. A single
injection of STZ (150 mg/kg body weight, i.p.) induced diabetes in both
male WT and KO mice. However, diabetes in KO mice (KO+STZ)
progressed rapidly to severe conditions with blood glucose reaching
or exceeding 600 mg/dL, resulting in a high mortality rate within two
weeks of treatment (Figure 1). On the other hand, diabetes in WT mice
(WT+STZ) was much less severe and did not cause death during the
two-week treatment. The average glucose levels for control WT and KO
mice were similar to each other (WT, 138.7 ± 17.5 mg/dL; KO, 139 ±
13.8 mg/dL); the average for WT+STZ was 380 ± 17.3 mg/dL (p<0.001,
n=8, compared with WT); and the average for KO+STZ was 558 ± 38.3
mg/dL (p<0.001, n=8, compared with WT; p<0.01, n=8, compared with
WT+STZ). Both WT and KO control mice gained body weight slightly
by ~0.25 to 0.3 grams during the two-week experiment; whereas, STZtreated
WT and KO mice lost body weight by an average of 1.5 or 2.1
grams, respectively. STZ may cause tissue damage independently of pancreatic islet b cell lesions, which may have contributed to increased
toxicity observed in diabetic KO mice. Therefore, additional groups
of WT and KO mice were given STZ injection followed by insulin to
maintain the blood glucose level within a normal range. These mice
survived well and did not develop diabetes in either WT or KO groups
(Figure 1B, WT+STZ+Insulin and KO+STZ+Insulin). Thus, the
mortality in diabetic KO mice was caused by diabetes. These results
revealed that KO mice are highly sensitive to STZ-induced diabetes and
diabetic lesions compared with WT mice.

In order to better assess the role of Nrf2 in diabetic myocardial
lesions in KO mice, diabetes was induced by STZ and insulin was
supplemented to maintain blood glucose levels between 400 and 600
mg/dL. No lethality was observed in the mice, but diabetic KO mice
were generally weaker than diabetic WT mice causing, for instance,
a greater loss of body weight in diabetic KO than WT mice (data
not shown). Histological examination of the mice revealed severe
pathological changes in the hearts of diabetic KO mice in comparison
with those of control KO and diabetic WT mice. As shown in Figure
2A, control hearts had regular and intact myocardial arrangements and
clearly visible nuclei (a, d, g, and j). Diabetic WT hearts (WT+STZ,
b and h) showed focal cell death and certain irregularity of the
myocardial fibers. On the other hand, diabetic KO hearts from both
male and female mice (KO+STZ; e and k) exhibited large areas of
irregular myocardial arrangements, myofibrillar discontinuation,
and cell death (karyorrhexis, pyknosis, and karyolysis). WT and KO
mice injected with STZ followed by insulin (STZ+Insulin) exhibited a
normal morphology of myocardial structures (c, f, i, and l), indicating
that the above observed pathology was not due to a direct toxicity of
STZ on the heart but reflected a diabetic complication in the heart.
Ultrastructurally, reduced density, discontinuation of myocardial
fibers and altered morphology of mitochondria were observed in the
hearts of both WT and KO diabetic mice. However, the lesions were
more diffuse and severe in KO than WT mice (Figure 2B). Additionally,
more non-myocardial cells were seen in the cardiac tissues of diabetic
KO mice than in control KO and diabetic WT mice.

The prominent structural lesions in diabetic KO mouse hearts
implicate impairment of myocardial functions. Therefore, we
examined the contractility of cardiomyocytes of both WT and KO mice
with or without diabetes. Fourteen days after STZ injection, AMVM
were isolated from mouse heart and were stimulated for contraction
with the β-adrenergic agonist isoproterenol. As shown in Figure 3,
AMVM from control KO mice showed significantly lower contractile
force (expressed as isoproterenol-evoked shortening) than AMVM
from control WT mice (i.e., ~25% of WT), revealing that loss of Nrf2
reduced the contractility of cardiomyocytes in normal mouse heart.
Diabetes decreased isopreterenol-evoked contraction of WT AMVM
by ~30% as compared with WT control. On the other hand, AMVM
from diabetic KO mice showed nearly a total loss of responsiveness
to isoproterenol for contraction, indicating diabetes severely impaired
the contractile function of KO hearts compared with WT, which was
in agreement with the structural changes described above. Taken
together, the structural and functional analyses revealed that loss of
Nrf2 caused severe cardiomyopathy under diabetic conditions.

Because cardiomyocytes in adult hearts are non-dividable,
apoptosis of the cells would result in a loss of myocardial tissues and
contractility. To examine if apoptosis of cardiomyocytes contributes
to the apparent pathological lesions and functional impairment in diabetic KO hearts, TUNEL assay was performed. Very few apoptotic
cells were present in non-diabetic, WT hearts (Figure 4A). Diabetes
induced a slight increase in the number of apoptotic cells in WT hearts.
In control KO hearts, apoptosis was slightly higher than that of control
WT hearts. On the other hand, apoptosis in diabetic KO hearts was
markedly increased indicating that diabetes substantially increased
apoptosis in the hearts of KO mice. Cells undergoing apoptosis have
increased cleavage of PARP1. Immunoblotting of PARP1 cleavage
product confirmed that diabetes induced apoptosis in WT hearts
(Figure 4B). Moreover, control KO hearts exhibited elevated PARP1
cleavage to the level comparable to that of diabetic WT hearts
indicating markedly elevated spontaneous apoptosis in mouse heart in
the absence of Nrf2. Unlike the findings from TUNEL assay described
above, PARP1 cleavage in diabetic KO hearts was not higher than but
similar to that in control KO hearts; this difference between PARP1
cleavage and Tunnel assay could be due to differential sensitivities of
the two assays to severe tissue damage in diabetic hearts. Nonetheless,
the findings demonstrated a critical role of Nrf2 in protection against
apoptosis in mouse heart under both basal and diabetic conditions.

Myocardial lesions in diabetes stimulate inflammatory responses
that are important for the clearance and repair of damaged tissues. However, in many cases, inflammation could worsen the pathological
lesions resulting in, for instance, fibrosis in the heart. Indeed, we
found that, in agreement with the severity of pathological lesions in
the heart, granulocyte infiltration was observed in diabetic hearts,
but the inflammatory response was significantly more pronounced
and spread-out in the hearts of Nrf2 KO than WT mice (Figure 5A and B). Similarly, diabetes stimulated macrophage infiltration in the
hearts, which was significantly more evident in KO than WT (Figure
5C and D). The findings implicate Nrf2 as a critical regulator of the
inflammatory response in diabetic hearts.

To examine fibrotic lesions, heart sections were stained for collagen
fiber formation (Figure 5E and F). Fibrosis was barely detectable in
non-diabetic hearts of male and female WT mice. Diabetes significantly
increased collagen fiber staining in WT hearts. On the other hand, fibrosis was clearly observed in control hearts of both male and female
KO mice. Fibrosis in KO hearts was drastically increased by diabetes as
compared with control KO and diabetic WT hearts of both male and
female mice. Increased fibrosis in diabetic KO hearts indicated that the
diabetic damage in KO hearts occurred earlier and more severely than
that in WT hearts in both genders.

Heightened oxidative stress

High levels of blood glucose could damage the heart by causing
oxidative stress in cardiomyocytes. On the other hand, Nrf2 protects
against oxidative stress by suppressing ROS production and reducing
oxidative damage in a range of cell types and tissues. Therefore, high
blood glucose and loss of Nrf2 function in diabetic KO mice could
synergistically enhance oxidative stress in the hearts, which in turn
causes severe cardiomyopathy. To directly test this possibility, we
measured oxidative damage in the hearts. ROS are known to damage
macromolecules including DNA, lipids, and proteins in cells. 8-OHdG,
an oxidized nucleoside of nuclear and mitochondrial DNA, is the most
commonly detected DNA lesion by ROS. We found that control WT
mice had a low background of 8-OHdG in the heart, whereas control
KO mice showed a significantly higher level of 8-OHdG in the heart
indicating lack of Nrf2 promoted oxidative stress even in the absence
of diabetes (Figure 6A). Diabetes significantly increased the amount of
8-OHdG in WT hearts supporting the notion that high glucose induces
myocardial oxidative stress. On the other hand, 8-OHdG in diabetic
KO hearts was substantially higher than that of either diabetic WT or
control KO hearts, demonstrating a heightened oxidative stress state in
the heart with combined high glucose and Nrf2 null function.

Diabetes significantly increased lipid peroxidation as measured
by increased formation of free MDA, a marker of lipid peroxidation,
in the hearts of WT mice (Figure 6B). In control KO mice, MDA
was significantly higher than that of the control WT mice. Diabetes
markedly increased MDA production in the KO hearts, which was
higher than either control KO or diabetic WT hearts. Thus, oxidative
lipid damage measured by MDA in the hearts was in agreement with
oxidative DNA damage measured with 8-OHdG.

Oxidative stress is often accompanied with increased nitrosative
damage to proteins from reactive nitrogen species (RNS). Nitrosative
protein damage can be measured as increased formation of 3-NT
in tissues, one of the most common markers of nitric oxide (●NO)-
dependent oxidative damage. Immunoblotting of 3-NT in the hearts
revealed that STZ injection induced formation of 3-NT in WT mice
(Figure 6C and 6D), but the highest amount of 3-NT was observed
in diabetic KO mice. The results indicated that increased nitrosative
damage occurred in diabetic cardiac tissues and that Nrf2 protected the
heart from nitrosative damage.

Given the important role of Nrf2 in protection against oxidative
damage, the heightened cardiac oxidative stress and inflammatory
changes indicate that lack of Nrf2 in the heart resulted in intrinsic
lesions in cellular oxidative defense that synergize with hyperglycemia
to cause the severe myocardial lesions. To analyze the mechanism
of action of Nrf2 in protection against diabetic cardiomyopathy at a
molecular level, transcriptional regulation of the cytoprotective genes
in the heart was examined. Nrf2 mRNA was expressed in the hearts
of the WT but not KO mice (Figure 7) because a major portion of the
Nrf2 gene was not transcribed in Nrf2 KO cells. Nqo1 is a prototype
of Nrf2-controlled, ARE-dependent gene encoding the cytoplasmic
detoxification enzyme NQO1. Nqo1mRNA was detected in WT but
not Nrf2 KO hearts confirming that Nrf2 is required for the basal expression of the gene in the heart. Diabetes significantly induced Nqo1
mRNA expression in WT hearts. However, induction of Nqo1 was lost
in Nrf2 KO mice. Expression of heme oxygenase 1 (Ho1), another
Nrf2 and ARE-dependent gene, mRNA followed a similar pattern to
Nqo1 (data not shown). Thus, loss of Nrf2 disrupted the protective,
transcriptional response to oxidative stress in diabetic heart to promote
oxidative damage.

Figure 7: Induction of cytoprotective genes. Total RNA was prepared from
hearts of control and diabetic Nrf2 WT and KO mice. Expression of mRNA of
Nqo1, Nrf2, and Action was expressed as fold change over control.**, p<0.01.

Discussion

Diabetic cardiomyopathy can occur clinically in the absence of
coronary artery disease and hypertension and thus, is recognized
as a distinct diabetic complication that contributes significantly to
heart failure among diabetic patients [2-4]. Specific therapeutic and
preventive measures for diabetic cardiomyopathy are currently lacking
[32]. The mechanism by which high blood glucose damages myocardial tissues to cause the disease and the factors that influence this pathogenic
process remain elusive. We have previously reported that high glucose
induced elevated production of ROS in cultured neonatal and adult
ventricular myocytes, which correlated with increased apoptosis and
mitochondrial damage [10]. These findings support the notion that
oxidative damage is a major contributor to high glucose-induced
myocardial lesions in diabetes [5-9]. Presumably, a hyperglycemiainduced
process of overproduction of superoxide by the mitochondrial
electron-transport chain leads to multiple molecular and biochemical
changes in cells and tissues that may involve the activation of multiple
signaling pathways [5,7,33]. Such changes would also include the
activation of cellular defensive mechanisms against oxidative stress and
tissue damage important in the development of diabetic lesions.

In the previous study, we found that Nrf2, an oxidant/antioxidant/
electrophile-activated receptor/transcription factor, is important
in defense against high glucose-induced oxidative damage in cardiomyocytes [10]. Cardiomyocytes isolated from Nrf2 KO mice
had significantly higher levels of ROS production than WT controls
under basal conditions and high glucose (20 to 40 mM) increased ROS
production, which was significantly higher in KO than WT cells. Parallel
to increased oxidative stress, the KO cells had a higher percentage
of cells undergoing apoptosis, increased sensitivity to mitochondrial
damage by mitochondrial complex II inhibitor 3-nitropropionic acid,
reduced response to β-agonist for contraction, and decrease or total
loss of induction of certain cytoprotective enzymes and proteins upon
stimulation with high glucose. The current study was designed to
examine if Nrf2 is important in protection against diabetic myocardial
lesions in intact animals. Our results demonstrated that Nrf2 KO mice
were highly sensitive to STZ-induced diabetic cardiomyopathy, which
resulted from heightened oxidative stress in the heart of diabetic KO
mice.

Nrf2 has been implicated in a wide range of diseases, such as
cancer and chronic degenerative diseases, and toxicities, such as
metal toxicity, polycyclic aromatic hydrocarbon-induced cancer, and
chemical/particle/fiber-induced lung fibrosis [18-24,34]. A common
theme emerged from the diseases was that they all have oxidative stress
as a component in disease pathogenesis and Nrf2 appears to suppress
ROS production and oxidative lesions in various experimental systems.
In this study, we found that loss of Nrf2 caused markedly increased
oxidative DNA damage, lipid peroxidation, and protein nitrosation
in the heart of diabetic KO mice, all indicating exacerbated oxidative
stress in the heart.

Three mechanisms may account for increased oxidative damage
in Nrf2 KO hearts. First, loss of Nrf2 in the heart resulted in the loss
or reduction of the expression and induction of ARE-dependent
cytoprotective genes including NQO1 and HO1. Furthermore, we
found that the expression of a number of enzymes important in
ROS metabolism including glutathione peroxidase 2, 6, and 8 were
down-regulated, whereas expression of several enzymes that promote
ROS production was increased in the Nrf2 KO heart (He and Ma,
unpublished observation). These findings are consistent with our
previous conclusion that loss of Nrf2 caused intrinsic defect in the
defense against oxidative damage in cardiomyocytes due to the lack
or reduced expression of ARE-dependent genes. Second, STZ induced
higher levels of glucose in KO mice than in WT mice, raising the
possibility of increased sensitivity of pancreatic islet β-cells to STZinduced
apoptosis in Nrf2 KO mice; further studies are underway
to analyze the role of Nrf2 in β-cell function. Third, cardiomyocytes
are prone to damage by ROS due to their continuous and repetitive
contraction for pumping blood and high energy demand from
mitochondrial respiration, both of which may lead to increased ROS
production. Therefore, the high sensitivity of the KO heart to diabetes
likely reflects a synergistic effect among hyperglycemia, Nrf2 null
function, and vulnerability of cardiomyocytes to ROS, which leads to a
heightened oxidative stress state and structural and functional lesions
in the heart.

We observed significantly increased infiltration of neutrophils
and macrophages in diabetic KO hearts. Furthermore, collagen fiber
formation, an indication of fibrosis that generally occurs during
chronic tissue damage, was markedly elevated as compared with
WT, suggesting that myocardial lesions in KO hearts occurred earlier
and more severely than in WT. Increased inflammatory response is
commonly seen in animal models of disease and toxicity with Nrf2
knockout [18]. Conversely, many Nrf2 activators potently inhibit
inflammation in multiple tissues [35]. Mechanistically, Nrf2 may suppress inflammation via two separate, but not mutually exclusive,
mechanisms: (a) inhibition of oxidative damage to tissues, which
stimulates inflammation; and (b) inhibition of the intrinsic functions of
inflammatory cells. In the latter case, Nrf2 may regulate inflammatory
and immune function by controlling the transcription of inflammation
and immune-specific genes. Further studies are needed to distinguish
the possibilities.

Both Nrf2 and its associated protein Keap1 are redox sensors.
Studies on ligand-Keap1 interaction by mass spectrometry and on
Keap1 structure by crystallography, NMR, and single particle electron
microscopy provided significant insights into the mechanism by
which Keap1 and Nrf2 interact with electrophilic inducers to activate
Nrf2 [28]. In this model, Nrf2 is constantly degraded with a half life
of only 20 min under basal conditions [25]. Degradation is mediated
through the Keap1/Cul3 dependent E3-controlled ubiquitination and
subsequent proteasomal degradation of Nrf2 [16]. Keap1 contains
~25 cysteine residues, whereas Nrf2 has 7 highly conserved cysteine
residues [26,27]. Electrophilic inducers interact with the thiol groups of
critical cysteine residues in both Keap1 and Nrf2 to trigger inhibition of
Nrf2 ubiquitination and degradation. Stabilized Nrf2 translocates into
the nucleus and mediates the basal and induced expression of target
genes through ARE-dependent transcripton. The mechanism by which
oxidants, such as ROS, activate Nrf2 is not clear. It is believed that ROS
and other oxidants oxidize critical cysteine residues of Keap1 and Nrf2
to activate Nrf2. Alternatively, substances, such as prostaglandins,
produced from oxidative and inflammatory tissue damage interact with
Keap1 and Nrf2 to activate Nrf2 analogously to electrophilic inducers.

Induction of ARE-controlled cytoprotective genes by natural
and synthetic antioxidants has been shown to be effective for the
prevention of cancer and certain chronic diseases for several decades
[15]. Recent discoveries of highly potent inducers, such as the synthetic
oleanane triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-
oyl] imidazole (CDDO-Im), opened new opportunities for disease
prevention and therapy [35]. For instance, CDDO-Im has a CD value of
0.0033 μmol/L (concentration required to double the specific enzyme
activity of NQO1), and has been shown to effectively inhibit aflatoxininduced
hepatic tumorigenesis [34], vinyl carbamate-induced lung
tumors [36], and cigarette smoke-induced emphysema and cardiac
dysfunction [37]. Given that diabetic cardiomyopathy is a chronic
complication of diabetes and no effective prevention and therapy are
available, the current finding that Nrf2 plays a critical role in protection
against diabetic myocardial lesions suggest that Nrf2 activators, such
as CDDO-Im, can be expoited for the prevention and treatment of the
disease in the future.

Acknowledgements

The findings and conclusions in this report are those of the authors and do not
necessarily represent the views of the National Institute for Occupational Safety
and Health.